Renal Failure, 30:843–847, 2008 Copyright © Informa Healthcare USA, Inc.ISSN: 0886-022X print / 1525-6049 onlineDOI: 10.1080/08860220802353827

843

LRNFCLINICAL STUDY

Chemiluminescent Analysis of Plasma Antioxidant Capacity in Uremic Patients Undergoing Hemodialysis

Chemiluminescent Analysis of Uremic PlasmaTung-Sheng ChenDepartment of Biomedical Engineering, Chung Yuan Christian University, Chung Li, Taiwan 32023, R.O.C

Shaw-Yih LiouFormosan Blood Purification Foundation, Taipei, Taiwan 10058, R.O.C

Yen-Lin ChangDepartment of Biomedical Engineering, Chung Yuan Christian University, Chung Li, Taiwan 32023, R.O.C

Objective. Hemodialysis is a common therapeutic strategyfor patients with end stage renal failure. During the hemodialyticprocess, the neutrophils are activated (neutrophil burst) due to thehemoincompatibility induced by hemodialysis. As a result, theactivated neutrophils release reactive oxygen species (ROS),such as hydrogen peroxide, singlet oxygen, and hypochlorite, intothe bloodstream and cause oxidative damage. Methods. Thisstudy investigated the antioxidant alteration of plasma in uremicpatients undergoing hemodialysis by chemiluminescent analysis.The antioxidant capacities of plasma in scavenging hydrogenperoxide, singlet oxygen, and hypochlorite were investigated inthis experiment. In addition, investigation of the ferric-reducingability of plasma (FRAP) would be covered in this study aswell. Results. This study found that after hemodialysis, theantioxidant capacities of plasma in scavenging hydrogen perox-ide, singlet oxygen, and hypochlorite decreases 7.9%, 18.8%,and 18.9%, respectively. Moreover, the FRAP is reduced by 56%.We speculate that the loss of dialyzable solutes (such as uremicsolutes and antioxidants with small molecular weight) in plasmaresulted in its decrease in antioxidant capacity. Conclusion. Wetherefore suggest that the supplement of antioxidants withsmall molecular weight is capable of regaining antioxidant

defense in plasma and preventing oxidative damage induced byhemodialysis.

Keywords hemodialysis, hemocompatibility, neutrophil burst,oxidative stress, FRAP, chemiluminescence

INTRODUCTION

Hemodialysis is a common therapeutic strategy forpatients at the end stage of renal failure. Althoughhemodialysis is capable of removing metabolic wastes(uremic toxins) through diffusion, it causes the imbal-ance of homeostasis due to the hemoincompatibilityinduced by the contact between blood and artificialhemodialyzer. The production of oxidative stress is oneof the results induced by hemoincompatibility.[1,2] Whenplasma oxidative stress is elevated, the risk of complica-tions associated with oxidative stress, such as atheroscle-rosis, inflammation, immunodysfunction, and cancer, is alsoincreased.[3,4]

The most acceptable mechanism for the productionof oxidative stress induced by hemoincompatibility is“neutrophil burst.”[5,6] The hemoincompatibility inducedby hemodialysis activates the neutrophils, which then releasereactive oxygen species (ROS) such as hypochlorite,hydrogen peroxide, and singlet oxygen into the blood-stream. These ROS can react with plasma biomolecules(such as proteins, lipids, and cell membrane materials) andcause oxidative damage on these biomolecules. As such,the monitor of alteration of oxidative stress in uremicpatients becomes an important issue during hemodialysis.

Received 12 March 2008; revised 14 July 2008; accepted 19July 2008.

Address correspondence to Prof. Yen-Lin Chang, Departmentof Biomedical Engineering, Chung Yuan Christian University,200, Chung Pei Rd., Chung Li, Taiwan 32023, R.O.C; Tel.: 8863 2654501 Ext. 4505; Fax: 886 3 2654505; E-mail: g9302504@cycu.edu.tw, yenlin@cycu.edu.tw

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The determination of oxidative metabolites in plasma(such as malondialdehyde, protein carbonyls, and oxidizedLDL) is the most common method in monitoring the alter-ation of oxidative stress in uremic patients undergoinghemodialysis.[7,8] This is primarily because the high con-centration of oxidative metabolites in plasma indicates ahigh risk of oxidative damage.

Another method in determining oxidative stress inuremic patients is to investigate the alteration of plasmaantioxidant capacity, including the ferric-reducing ability ofplasma (FRAP) or its oxygen radical absorbance capacity(ORAC).[9,10] The decrease of plasma antioxidant capacityleads to a high risk in ROS attack. Moreover, chemilumines-cent analysis is also used in determining plasma oxidativestress.[11] The oxidants existing in plasma react with light-emitting materials (luminol or lucigenin), which then releaseultra-weak luminescence that can be detected by chemilu-minescent analyzer. The intensity of chemiluminescence isproportional to the concentration of plasma oxidants.

This study aims to investigate the alteration of plasmaantioxidant capacities in scavenging singlet oxygen,hypochlorite, and hydrogen peroxide (the three ROSmainly released from neutrophil burst) in uremic patientsundergoing hemodialysis by chemiluminescent analysis.This study also covers the antioxidant capacities of somefood extracts in scavenging the above-mentioned ROS bychemiluminescent analysis.

METHODS

Chemicals and Reagents

In this study, acetonitrile and ethanol were purchasedfrom Mallinckrodt Baker, Inc. (Phillipsburg, New Jersey,USA), while phosphate buffered saline (PBS) was purchasedfrom Invitrogen Corporation (Grand Island, New York,USA). Other chemicals and reagents used in this studywere from Sigma-Aldrich Inc. (St. Louis, Missouri, USA).

Experimental Subjects

Sixteen healthy volunteers and twenty uremic patientswere involved in this experiment. The uremic patients con-tained both genders (11 males and 9 females) with averagedhemodialytic duration for 89 ± 24 months. The hemodialyticprocesses for the uremic patients followed the standard opera-tion procedures (SOP) in Formosan Blood Purification Foun-dation (Taipei, Taiwan). Healthy volunteers (nine males andseven females) were selected according their laboratoryblood tests. The plasma samples collected from uremic andhealthy volunteers were stored at −80°C before use.

Singlet Oxygen-Scavenging Potential

The first step of this experiment was to produce singletoxygen in chemiluminescent analyzer by mixing Na2CO3buffer, H2O2 and acetonitrile in basic condition.[12] In theexperiment, 0.5 mL of Na2CO3 buffer (100 mM), 0.05 mLof H2O2 (1.7%), and 0.05 mL of diluted plasma oranalytes were added into the chemiluminescent analyzer(CLA-FS1, Tohoku Electronic Industrial Co. Ltd., RifuTown, Miyagi, Japan). After mixing for 20 seconds,0.1 mL of luminol solution (20 mg of luminol in 20 mLof 30% acetonitrile) was added by injection. Intensivechemiluminescence was detected in this step due to singletoxygen production. The chemiluminescent measurement wasterminated after 60 seconds. During the experiment, the dis-tilled water acted as blank and trolox (6–Hydroxy–2,5,7,8–tetramethylchroman–2–carboxylic acid, a water-solublevitamin E-like antioxidant) as standard. The scavengingpotentials of singlet oxygen for the analytes were expressedas trolox equivalents (TE).

Hypochlorite-Scavenging Potential

The experimental protocol was modified from Liet al.[13] In this experiment, 1 mL of distilled water, 0.02 mLof NaOCl (0.037%), and 0.1 mL of diluted plasma or ana-lytes were added into the chemiluminescent analyzer.After mixing for 20 seconds, 0.1 mL of luminol solution(44 mg of luminol in 10 mL of 0.01 N sodium hydroxide)was added by injection. Intensive chemiluminescence wasobserved in this step. The chemiluminescent measurementwas terminated after 90 seconds. During the experiment,the distilled water acted as blank and trolox as standard.The scavenging potentials of hypochlorite for the analyteswere expressed as trolox equivalents.

Hydrogen Peroxide-Scavenging Potential

The chemiluminescence induced by H2O2 can bemeasured by mixing H2O2 and lucigenin in tris buffer.[14]

In this study, 0.1 mL of H2O2 (1.7%), 1 mL of tris buffer(50 mM, pH = 7.2), and 0.1 mL of diluted plasma or ana-lytes were added into the chemiluminescent analyzer.After mixing for 20 seconds, 0.1 mL of lucigenin solution(1.96 mM) was added by injection. Intensive chemilumi-nescence was measured in this step. The chemilumines-cent measurement was terminated after 90 seconds.During the experiment, the distilled water acted as blankand trolox as standard. The scavenging potentials ofhydrogen peroxide for the analytes were expressed astrolox equivalents.

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Chemiluminescent Analysis of Uremic Plasma 845

Ferric-Reducing/Antioxidant Power (FRAP) Assay

The FRAP assay for this experiment was modifiedfrom Othman et al.[15] The FRAP reagent was prepared bymixing acetate buffer (246.1 mg of sodium acetate in 10 mLof 10% acetic acid), TPTZ solution (31.23 mg of TPTZ and0.044 ml of 37% HCl in 10 mL of distilled water), andFeCl3 solution (54.06 mg of FeCl3·6H2O in 10 mL ofdistilled water) in a ratio of 10:1:1. The FRAP reagent(0.5 mL), distilled water (0.05 mL), and 0.016 mL of analyteswere transferred into vials and then incubated at room tem-perature. After incubation for 4 minutes, the absorbance (λ =593 nm) was measured for all vials. The distilled water actedas blank, and the FRAP data for the analytes were expressedas FeSO4 equivalents.

Blood Test for Uremic Patients

The quantification of plasma uremic markers (includ-ing blood urea nitrogen, creatinine, uric acid, and β2-MG)was determined by IMMULITE instrument (DPC CirrusInc., Los Angeles, California, USA).

Statistical Analysis

All experimental data were expressed as mean ±standard deviation (SD). Data analysis was carried out byusing paired t-test. Statistical difference was to be consid-ered at the level of p < 0.05.

RESULTS

Hydrogen peroxide oxidizes lucigenin, which releasesultra-weak luminescence that is detected by the chemilumi-nescent analyzer. As such, the intensity of chemilumines-cence is proportional to the concentration of hydrogenperoxide. Moreover, the addition of plasma in hydrogen per-oxide-lucigenin reaction leads to a decrease in chemilumi-nescent intensity because the antioxidants existing in plasmascavenge hydrogen peroxide. Therefore, the reduction ofchemiluminescence positively correlates with plasma antiox-idant capacity in scavenging hydrogen peroxide. Figure 1shows the antioxidant capacity of plasma in scavengingchemiluminescence induced by hydrogen peroxide-lucigeninreaction. The antioxidant capacities of pre-hemodialytic(HD1) and post-hemodialytic (HD2) plasma samples are2.495 ± 0.452 TE and 2.298 ± 0.524 TE, respectively. Bycomparison of HD1 and HD2, the antioxidant capacity ofplasma in scavenging hydrogen peroxide is reduced by 7.9%(p < 0.05) in the uremic patients after hemodialysis.

Similar results can be observed in Figures 2 and 3.Figures 2 and 3 show the antioxidant capacities of plasmain scavenging chemiluminescence induced by hypochlo-rite and singlet oxygen, respectively. In Figure 2, theantioxidant capacities for HD1 and HD2 are 0.062 ± 0.009TE and 0.051 ± 0.011 TE. In Figure 3, the antioxidantcapacities for HD1 and HD2 are 0.682 ± 0.071 TE and0.554 ± 0.089 TE. The antioxidant capacities of plasma inscavenging hypochlorite and single oxygen are respectively

Figure 1. Hydrogen peroxide scavenging power of plasmacollected from different subjects.

Figure 2. Hypochlorite scavenging power of plasma collectedfrom different subjects.

Figure 3. Singlet oxygen scavenging power of plasma collectedfrom different subjects.

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reduced 18.9% (p < 0.05) and 18.8% (p < 0.05) in uremicpatients after hemodialysis.

In addition to chemiluminescent analysis, we also investi-gated the FRAP in uremic patients with hemodialysis. TheFRAP assay evaluates the electron-donating ability of plasma.Antioxidants are known for easily donating electrons to termi-nate chain reactions induced by free radicals. This is one rea-son why antioxidants are capable of scavenging free radicals.Therefore, the FRAP value is proportional to the antioxidantcapacity of plasma. In Figure 4, the FRAP values for HD1 andHD2 are 827 ± 161 and 363 ± 109, respectively. This meansthat the plasma FRAP (antioxidant capacity) has decreased56% (p < 0.05) in uremic patients after hemodialysis.

Table 1 shows the results of blood test for uremicpatients undergoing hemodialysis. We can observe theclearance percentage of uremic solutes including bloodurea nitrogen (73.93%), creatinine (69.67%), uric acid(79.63%), and β2-MG (55.44%).

Table 2 shows the chemiluminescent analysis of somefood extracts in scavenging hydrogen peroxide, hypochlo-rite, and singlet oxygen. It indicates that the green tea extract(GTE) exhibits the most excellent capacities in scavengingabove three ROS compared to the apple extract (AE),Rhodiola rosea extract (RE) and Eleutherococcus senticosisextract (EE). In addition, Figure 5 shows the FRAP assay for the four extracts. In high concentration (0.1 mg/mL),

the FRAP of the four extracts follow the order of GTE(897 ± 37) > RE (402 ± 16) > EE (121 ± 11) > AE (13 ± 1).In low concentration (0.01 mg/mL), the order of the FRAPfor the four extracts is GTE (138 ± 20) > RE (66 ± 3) > EE(undetectable) ≅ AE (undetectable).

DISCUSSION

Hydrogen peroxide, hypochlorite, and singlet oxygenare the main ROS released from neutrophil burst induced byhemodialysis. Based on our experimental results, the anti-oxidant capacities of plasma in scavenging the above threeROS were decreasing in uremic patients after hemodialysis.We speculate that some antioxidant dialyzable solutes wereremoved during hemodialysis. For instance, uric acid isstudied to exhibit antioxidant property in plasma. In Table 1,we can find that the plasma level of uric acid depletes by79.63% after patients with hemodialysis. The loss of antiox-idant dialyzable solutes resulted in the defect of plasma anti-oxidant defense.[16–18] As such, this may be the reason whyhemodialysis leads to a decrease of plasma antioxidantcapacity in uremic patients. The alteration of plasma FRAPin Figure 4 also supports this explanation.

Results of the experiment point out that the reductionof plasma antioxidant capacities in scavenging the abovethree ROS is in the order of hydrogen peroxide (7.9%) >singlet oxygen (18.8%) ≅ hypochlorite (18.9%). The

Figure 4. The FRAP assay of plasma collected from differentsubjects.

Table 1 Blood test for uremic patients undergoing hemodialysis

HD1 HD2 Clearance %

Blood urea nitrogen (mg/ml)

69.94 ± 13.60 18.23 ± 7.35 73.93

Creatinine (mg/ml)

11.77 ± 2.84 3.57 ± 1.40 69.67

Uric acid (mg/ml)

8.10 ± 1.39 1.65 ± 0.62 79.63

β2-MG (mg/ml)

2.94 ± 0.85 1.31 ± 0.53 55.44

*Data were expressed as mean ± SD.

Table 2 ROS scavenging capacities of some food extracts

Singlet oxygen Hypochlorite Hydrogen peroxide

GTE 44.43 ± 2.08 16.45 ± 0.71 4.16 ± 0.07AE 4.42 ± 0.25 1.75 ± 0.07 3.3 ± 0.07RE 39.25 ± 1.43 3.33 ± 0.22 0.69 ± 0.03EE 4.68 ± 0.17 5.45 ± 0.24 0.05 ± 0.002

*Data were expressed as mean (n = 6) ± SD and trolox equiv-alents (mg of trolox/mg of extract).

Figure 5. The FRAP assay of some food extracts. *Data wereexpressed as mean (n = 3) ± SD.

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Chemiluminescent Analysis of Uremic Plasma 847

reduction of plasma antioxidant capacity in scavenginghydrogen peroxide is less than the other two ROS. This maybe associated with the catalase level in plasma. The catalase,an enzyme that converts hydrogen peroxide into water andoxygen in plasma, has a molecular weight of about 230 kDa.The hemodialytic process is not able to remove the solutewith such a large molecular weight (averaged pore size ofhemodialyzer is less than 66 kDa). Therefore, the catalaselevel in plasma does not alter dramatically in uremic patientsafter hemodialysis. On the other hand, there is no specificplasma enzyme in scavenging hypochlorite and singlet oxy-gen. Instead, the removal of the two ROS depends on smallantioxidants existing in plasma. This may be a reason why thealteration of antioxidant capacity in scavenging hydrogenperoxide is less than the other two ROS.

In the experiment, the chemiluminescent analysis ofthe antioxidant capacities in scavenging the above threeROS as well as the FRAP assay for some food extracts werealso investigated. Results show that GTE exhibits the mostexcellent capacities in scavenging the above three ROS andthe best FRAP compared to RE, AE, and EE. This may bebecause GTE contains a high level of polyphenol ingredi-ents (the order of polyphenol ingredients: GTE > RE > AE> EE, data not shown). Further, Hsu et al. [19] pointed outthat supplement of GTE reduced hemodialysis-enhancedproduction of ROS. These data support the idea that thesupplement of GTE may assist uremic patients in avoidingoxidative stress induced by hemodialysis.

In summary, the supplement of antioxidants with smallmolecular weight (such as GTE in this article) is capable ofreplenishing the lost dialyzable solutes in uremic patients dur-ing hemodialysis. This can reduce oxidative damage inducedby the neutrophil burst and eventually improve the quality oflife of uremic patients undergoing hemodialysis.

DECLARATION OF INTEREST

The authors report no conflicts of interest. The authorsalone are responsible for the content and writing of the paper.

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